Nephropathy-associated gene

ABSTRACT

A nephropathy-associated gene which encodes a transcription repressor; and a nonhuman transgenic animal suffering from nephropathy which is constructed by transferring the above gene and allows the observation of increases in urinary volume, urinary albumin and urinary NAG, pyelectasis, enlargement in kidney tubule and glomerular swelling at the early stage and the following sclerosis.

TECHNICAL FIELD

The present invention relates to a nephropathy-associated gene, atransgenic non-human mammalian animal constructed by transferring thegene, a therapeutic agent for nephropathy, and a method for screeningmedicines effective for diagnosis or treatment of the nephropathy. Theinvention relates particularly to diabetic nephropathy in thenephropathy.

BACKGROUND ART

Nephropathy, for which no specific medicine is available, requires anartificial dialysis treatment in an advanced stage even though thistreatment inflicts pain on patients. In particular, diabetic neuropathyhas become a serious problem as the number of patients with diabetes hasincreased.

With regards to mechanisms of onset of diabetes, it is well known thatsevere insulin dysfunction induces a diabetic condition. Recently, oneof the mechanisms for insulin dysfunction has been elucidated (Inada A.et al., Bunshi Tonyobyogaku [Molecular Diabetology] 10:73-81, 1999), andelucidation of the onset mechanism of and studies on the treatment ofdiabetes have been actively performed. However, for mechanisms of onsetdiabetic complications involving diabetic nephropathy, diabeticretinopathy and diabetic mental disorder, there is no effective andestablished diagnostic or therapeutic management.

Meanwhile, to develop diagnostic or therapeutic management of diabetesor diabetic complications, vigorous effort has made to establish optimalanimal models. (JP 04-248941 A). However, animal model so far exhibitsonly hyperglycemia with low insulin but not diabetic complicationsidentical to that of human. It has been reported that only a doubletransgenic (Tg) mouse obtained by crossing two gene-modified Tg miceexhibits diabetic nephropathy (J. Clinical Investigation, Vol. 108, No.2, p. 261, 2001). However, in this double Tg mouse model, to developdiabetes and to maintain hyperglycemia, it is required to cross a Tgmouse that specifically overexpress inducible Nitric Oxide (iNOS) ininsulin-producing β cells (J. Biol. Chem., Vol. 273, pp.2493-2496, 1998)with a Tg mouse overexpressing receptor for advanced glycosylationendproduct (RAGE) in vascular cells. High proteinuria andglomerulosclerosis which are similar to human diabetic nephropathyappear only when both transgenes are simultaneously expressed. Inaddition, modified overexpressions of iNOS in pancreatic β cells andRAGE in vascular cells are unusual situation. Thus, this model isartificial and far from actual diabetic condition, and no single animalmodel develops renal changes identical to those seen in humans.

It has been known that ICER suppresses insulin (A. Inada et al JBC 1999vol. 274 no. 30 p. 21095-21103; A. Inada et al BBRC 1998 vol. 253 no. 3p. 712-718) gene transcription, and that ICER is increased in thediabetic condition (Inada A. et al BBRC 1998 vol. 253 no. 3 p. 712-718),but no association of ICER with diabetic nephropathy has been known.

Thus, the development of a medicine effective for the diagnosis or thetreatment of nephropathy including diabetic nephropathy and thedevelopment of a useful experimental system as a pathogenic model ofnephropathy or diabetic nephropathy have been highly required.

The present invention mainly intends to provide a method useful for thediagnosis and the treatment of nephropathy including diabeticnephropathy and a transgenic non-human mammalian animal useful as apathogenic model of diabetic nephropathy.

DISCLOSURE OF INVENTION

The present inventor carried out extensive studies mainly aiming atsolving the above subject matters. As a result of making a transgenicmouse (ICER Iγ Tg mouse) in which both insulin synthesis and β-cellproliferation are inhibited and after performing extensive studies, thepresent inventor has found that the ICER Tg mouse develops diabetes thatstably expresses major clinical and pathological features of humandiabetic nephropathy, and has completed the present invention by furtheranalyses.

That is, the present invention relates to the followings.

[1] A nephropathy-associated gene characterized by encoding an insulintranscription repressor belonging to a transcription factor CREM family.

[2] The gene according to [1] wherein the insulin transcriptionrepressor is any one selected from the group consisting of CREMα, ICER Iand ICER Iγ.

[3] The gene according to [1] wherein the insulin transcriptionrepressor is ICER Iγ.

[4] The gene according to [1] having a base sequence represented by SEQID NO:1, a complementary sequence thereof or a nucleotide sequence whichhybridizes therewith under a stringent condition, and capable ofdeveloping nephropathy.

[5] The gene according to [1] having a base sequence represented by SEQID NO:2, a complementary sequence thereof or a nucleotide sequence whichhybridizes therewith under a stringent condition, and capable ofdeveloping nephropathy.

[6] The gene according to [1] wherein the nephropathy is diabeticnephropathy.

[7] The gene according to [1] wherein the above gene is derived fromhuman.

[8] A nephropathy developing agent comprising the gene according to anyone of [1] to [7].

[9] A diabetic nephropathy developing agent comprising the geneaccording to any one of [1] to [7].

[10] A probe for determination or diagnosis of human nephropathycomposed of 15 base or more nucleotide sequence capable of hybridizingwith the gene according to [7] under a stringent condition.

[11] A nephropathy-associated protein having an activity of an insulintranscription repressor belonging to a transcription factor CREM family.

[12] The nephropathy-associated protein according to [11] which is anyone selected from the group consisting of CREMα, ICER I and ICER Iγ.

[13] The protein according to [11] derived from human.

[14] An antibody which specifically binds to the protein according toany of [11] to [13] or a part thereof.

[15] The antibody according to [14] which specifically binds to a humannephropathy-associated protein.

[16] A diagnostic medicine of human nephropathy containing the antibodyaccording to [15].

[17] An antisense DNA for the human nephropathy-associated geneaccording to [7].

[18] A preventive or therapeutic agent of neuropathy containing theantibody according to [15] or the antisense DNA according to [17] as anactive component.

[19] A method for determining or diagnosing nephropathy having a step ofexamining an expression level of the human nephropathy-associated geneaccording to [7] in a specimen using the probe according to [10] or theantibody according to [12].

[20] A transgenic non-human mammalian animal introducing the geneaccording to any one of [1] to [6].

[21] The mammalian animal according to [20] that develops nephropathy.

[22] The mammalian animal according to [21] wherein the nephropathy isdiabetic nephropathy.

[23] The mammalian animal according to [20] that develops diabetes.

[24] The mammalian animal according to [23] wherein the diabetescombines with diabetic nephropathy.

[25] A method for making a transgenic non-human mammalian animal havinga step of introducing the gene according to any one of [1] to [6].

[26] A method for screening a preventive or therapeutic agent for atleast one selected from the group consisting of nephropathy, diabeticnephropathy and diabetes, having a step of administering a subjectsubstance to the transgenic non-human mammalian animal according to anyone of [20] to [25] and measuring an effect of the subject substance onat least one selected from the group consisting of nephropathy, diabeticnephropathy and diabetes of the mammalian animal.

[27] An expression vector capable of stably expressing in mammaliancells, incorporating a marker gene under the control of a promoter ofthe human nephropathy-associated gene according to [7].

[28] A mammalian cell transformed with the expression vector accordingto [27].

[29] The cell according to [28] wherein the mammalian cell is a humancell.

[30] A method for screening a preventive or therapeutic agent for atleast one selected from the group consisting of human nephropathy,diabetic nephropathy and diabetes, comprising a step of culturing themammalian cells according to [28] or [29] in the presence of a medicinecandidate compound in culture of the mammalian cells and a step ofmeasuring an amount of the expressed marker gene in the presence orabsence of the candidate compound.

[31] A preventive or therapeutic agent for at least one selected fromthe group consisting of human nephropathy, diabetic nephropathy anddiabetes, containing a substance obtained by the method for screeningaccording to [26] or [30] as an active component.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows ICER Iγ expression and effects thereof produced in ICER IγTg mice. FIG. 1 a shows a produced construct. FIG. 1 b shows a detectionresult of ICER Iγ mRNA. The ICER Iγ mRNA was correctly expressed fromthe transgene construct in ICER Iγ Tg mice. FIG. 1 c shows proteinexpression of ICER Iγ detected by Western blotting. The protein, at alarge amount, was expressed in ICER Iγ Tg mice. FIG. 1 d shows insulinmRNA amounts. The insulin mRNA was scarcely expressed in ICER Iγ Tgmice.

FIG. 2 shows blood glucose levels and variations of blood parameters inorder to evaluate the effects of ICER Iγ expression. FIG. 2 a shows thechanges of the blood glucose levels, and white and black circlesindicate wild type (WT) and ICER Iγ Tg mice, respectively. FIG. 2 ashows the changes on day 0 and day 7. The blood glucose level wasincreased in ICER Iγ Tg mice on the 7th day after birth. FIG. 2 b showsthe changes of the blood glucose levels for 20 weeks. Tg mice developsevere diabetes as early as 2 weeks of age and sustain hyperglycemia.FIG. 2 c shows the changes of plasma insulin levels. Insulin synthesiswas suppressed in ICER Iγ Tg mice. FIG. 2 d shows the changes of ketonebodies (acetone, acetoacetic acid and β-hydroxybutyric acid). The levelsof the ketone bodies were increased in ICER Iγ Tg mice. FIG. 2 e showsthe changes of glucagon levels. Production of glucagon was increased inICER Iγ Tg mice. FIG. 2 f shows pancreatic islets stained withanti-insulin antibody (green) and anti-ICER Iγ antibody (red) in mice onthe 7th day after birth. In ICER Iγ Tg mice, the expression of ICER Iγand the decrease of insulin-producing cells were observed (a barrepresents 10 mm). One or two cells are shown in photographs at theright side.

FIG. 3 shows the change of body weight of ICER Iγ Tg and WT mice. FIG. 3b shows the changes on 0 day and the 7th day after birth. FIG. 3 a is aphotograph of a male Tg mouse and control littermate (WT) at 12 weeks ofage. FIG. 3c show the growth curve of male mice from day 0 to 20 weeksof age. There was no difference in growth until 7 days after birth (FIG.3 b), but the growth of ICER Iγ Tg mice was suppressed around 6 weeks ofage (FIG. 3 c).

FIG. 4 a shows the changes of urinary volumes, and ICER Iγ Tg miceexhibited polyuria. FIG. 4 b shows the changes of urinary albuminexcretion. Proteinuria was increased in ICER Iγ Tg mice with aging. FIG.4 c shows the changes of urinary NAG which is an indicator of renaltubular disorder. All levels of the urinary volume, urinary albumin andurinary NAG were increased with aging in ICER Iγ Tg mice. All levels ofthe urinary volume, urinary albumin and urinary NAG were furtherincreased in ICER Iγ Tg mice compared with WT mice.

FIG. 5 shows the results obtained by giving HE staining to kidneyspecimens and observing by an optical microscopy. Enlargement of renalpelvis and renal tubule was observed in the Tg mice.

FIG. 6 shows the results obtained by giving PAS staining to kidneyspecimens and observing by an optical microscopy. Compared with the WTmice, glomerular hypertrophy was observed in the Tg mice at 4 weeks ofage and sclerosis thereof was observed at 12 weeks.

FIG. 7 shows glomerulus in the mouse at 36 weeks of age. Obviousglomerular sclerosis was observed in ICER Iγ Tg mice (FIG. 7 a, b, c).The glomerular hypertrophy was quantitatively examined, and consequentlythe hypertrophy was observed from 4 to 12 weeks of age (FIG. 7 d).

FIG. 8 shows immunohistochemical staining for collagen type IV. Theincrease of collagen type IV was observed in ICER Iγ Tg mice.

FIG. 9 shows the expression of the extracellular matrix in glomeruli byimmunohistochemical staining for collagen type IV or laminin. In both ofA and B, a left and a right indicate renal sections at 40 weeks of agein the WT and ICER Iγ Tg mice, respectively. C and D indicate scoresobtained by determining from the renal sections. In ICER Iγ Tg mice,collagen type IV and laminin were quantitatively increased.

FIG. 10 shows islet morphology in mice from line Tg23.

-   A: Pancreatic sections from day 0 to 36 weeks of age were    immunohistochemically stained using anti-insulin antibody (pink) and    anti-glucagon antibody (red). In ICER Iγ Tg mice, it seems that    insulin-producing cells (β cells) have been already decreased at    birth (day 0) and tissue of the islets is severely disorganized with    significantly increased glucagon-producing cells (glucagon +). Since    the β cells were sharply decreased at day 0 at which the blood    glucose level is normal, indicating that these changes result    directly from the increased ICER Iγ and are not due secondarily to    hyperglycemia. B: Reduced insulin-producing cells (arrow) in ICER Iγ    Tg mice at 7 day after birth (×1000). In ICER Iγ Tg mice, there is a    marked variation in the amount of insulin per cell (degranulation)    as shown by the insulin staining.-   C: Dual staining of glucagon (green) and IAPP (red) as a β-cell    marker was analyzed by confocal microscopy. In ICER Iγ Tg mice, β    cells were reduced in number and most of the islet was glucagons    positive. A few cells co-expressing IAPP and glucagons were present.    Because the insulin level is significantly decreased and    anti-insulin antibody cannot detect the P cell in ICER Iγ Tg mice,    IAPP was used as a β-cell marker to demonstrate the presence of    degranulated β cells.-   D: Scattered singlets-doublets of insulin-positive cells in ICER Iγ    Tg mice at 7 days of age, suggesting that neogenesis of β cells was    normally occurred (×400). Thus, in this Tg model, diabetes resulted    from a decreased number of β cells, further compounded by impaired    insulin expression in individual β cells.

FIG. 11 shows cyclin A expression and proliferation in islets at day 7of age (line Tg23). A: Dual staining of insulin (red) and Ki67 (green)as a marker of cell proliferation was analyzed by confocal microscopy.Ki67 protein was detected in many nuclei of insulin-positive cells in WTmice but in no insulin-positive cells in ICER Iγ Tg mouse. B: cyclin Aexpression in islets at day 7 of age. At day 7, normally a time ofactive cell proliferation, cyclin A protein was detected in most of thenuclei in islets in WT mice but in only a few islets of ICER Iγ Tg mice.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is described in more detail below.

Nephropathy-Associated Gene

The gene, according to the present invention, encodes an insulintranscription repressor belonging to the CREM (cAMP responsive elementmodulator) family which modulates transcription of an insulin gene bybinding to a regulatory transcription factor (cAMP responsible element[CRE]). The present inventor has elucidated that the gene is involved inthe onset mechanism and pathological progress of nephropathy.

The repressors of insulin gene transcription, belonging to thetranscription factor CREM family, include ICER I (inducible cAMP earlyrepressor I), ICER Iγ, CREMα and the like. Among them, ICER Iγ isparticularly useful.

The nephropathy-associated gene of the present invention widelyencompasses nephropathy-associated genes derived from mammalian animalssuch as human, rat, monkey, dog, rabbit, hamster, cattle and horse inaddition to genes derived from a mouse (SEQ ID NOS:1, 2, etc.), and alsoencompasses these genes or complementary chains thereof orpolynucleotides capable of hybridizing therewith under a stringentcondition. In particular, the gene derived from the mouse is useful forthe production of transgenic mice, and the gene derived from the humanis useful for diagnosis of human nephropathy, evaluation of potentialnephropathy in future and search of therapeutic agents for humanpatients with renal diseases.

Herein, the stringent condition refers to the condition where onlyspecific hybridization occurs and non-specific hybridization does notoccur. Such a condition is typically a state of about “1×SSC, 0.1% SDSat 37° C.”, preferably about “0.5×SSC, 0.1% SDS at 42° C.”, and morepreferably about “0.2×SSC, 0.1% SDS at 65° C.”. For example, in the caseof nephropathy-associated gene derived from the mouse, a DNA obtained bythe hybridization usually has high homology to a DNA represented by abase sequence described in SEQ ID NO: 1 or 2. The high homologyindicates a homology of 60% or more, preferably a homology of 75% ormore, and most preferably a homology of 90% or more. Furthermore, theDNA obtained by the hybridization or a nephropathy-associated proteinencoded by the gene has an effect to develop nephropathy.

The transcription repressor which belongs to the CREM family is aregulatory factor of insulin and cyclin A gene transcription activity.When this repressor is excessively expressed, the insulin production isreduced, and β-cell replication are inhibited. As a result, islets havereduced β-cell numbers and impaired insulin secretion, leading tochronic hyperglycemia, which results in severe diabetes. The presentinventor has elucidated that ICER Tg mouse model exhibits not onlysevere diabetes but also develops progressive diabetic nephropathy.These results suggest that the repressor and the gene encoding repressorare deeply involved in onset and pathological progress of nephropathy.

The nephropathy-associated genes of the present invention having suchproperties contribute to elucidate an onset mechanism and progress ofnephropathy, to further elucidate pathogenesis and treatment of patientswith nephropathy, and can be used usefully for nephropathy onset,analysis of nephropathy, detection of nephropathy, diagnosis ofnephropathy and the like.

In particular, when the gene of the invention is highly expressed,diabetic nephropathy develops. Thus, it is possible to usefully applyfor the onset, analysis, detection and diagnosis of diabeticnephropathy.

The gene of the present invention can be utilized as a nephropathydeveloping agent or a diabetic nephropathy developing agent byincorporating as needed in a vector and the like under the control of apromoter which is highly expressed in a mammalian animal that is a host.Nephropathy developing agent or diabetic nephropathy developing agentcan be utilized for the production of a model animal for diabeticnephropathy development.

The nephropathy-associated gene of the present invention develops thenephropathy (particularly, diabetic nephropathy), and is likely tosimultaneously develop diabetic complications (angiopathy, retinopathy,neuropathy etc.) other than nephropathy when the gene is expressed athigh levels. In particular, neuropathy is observed in an STZ(streptozotocin)-induced diabetes model, and thus, it is highly likelythat the neuropathy also occurs in the transgenic mice of the presentinvention.

The neuropathy-associated genes and GeneBank access numbers thereof areexemplified below:

-   AJ311667, AJ292222 (mouse ICER I)-   S67786 (mouse ICER Ir);-   AB031423 (rat CREM17X)-   S66024 (rat ICER II)-   U44836 (human ICER I)-   S68271, Z15159, Z15158 (human CREM)-   U04835 (rat CREM)-   M60285 (mouse CREM)

The neuropathy-associated genes of the present invention are not limitedthereto, widely encompass the insulin transcription repressors belongingto the CREM family, and further encompass modified genes capable ofhybridizing with such insulin gene transcription repressors under thestringent condition.

The neuropathy-associated gene of the invention can be expressedsystemically, but it is preferable to selectively express in organs suchas pancreas and kidney, preferably the pancreas, and more preferablyLangerhans islet cells in the pancreas. For example, selectiveexpression in the Langerhans islet cells can be accomplished byexpressing the nephropathy-associated gene under the control of aninsulin promoter. The selective expression in the other site/organ canbe accomplished by placing the nephropathy-associated gene of theinvention under a promoter of a gene to be selectively expressed in thesite/organ.

For example, to induce nephropathy (including the diabetic nephropathy)and diabetes models, it seems useful that expressing repressors underthe β-cell specific promoter, such as Pdx-1, IAPP, in addition to theinsulin promoter.

When the nephropathy-associated gene of the invention is expressed at ahigh level, levels of collagen IV and laminin are increased, and thesephenomena indicate glomerular sclerosis. When the glomerular sclerosisoccurs, a glomerular filtration rate (GFR) is reduced and renaldysfunction occurs to develop renal failure and finally uremia.Concomitantly with this, intravascular pressure is increased to develophypertension. Therefore, by expressing the nephropathy-associated geneat a high level, it is possible to produce pathological models forvarious renal diseases.

Probe

A probe of the present invention is composed of a sequence of 15 base ormore capable of hybridizing with the above gene of the invention underthe stringent condition, particularly composed of a nucleotide sequencecomplementary to the gene of the invention or a complementary chainthereof, and can be used usefully for the purpose of examiningnephropathy-related issues such as determination or diagnosis ofnephropathy. It is also possible to use for the analysis or detection ofnephropathy. In particular, the probe of the invention can be suitablyused for the determination or diagnosis of diabetic nephropathy.

In one preferable embodiment of the present invention, the probe has apart of the sequence of human nephropathy-associated gene subjected tothe diagnosis of the human nephropathy.

The probe of the invention can be also utilized as a disease marker byimparting an appropriate label.

The probe or the disease marker of the invention can be used effectivelyfor the diagnosis or determination of nephropathy or diabeticnephropathy such as examinations of presence or absence of the onset ofnephropathy or progression stages of nephropathy or diabetic nephropathyby using for a step of contacting with a specimen sample and measuringthe presence or absence of and an amount of a gene bound to the probe oran expression product (mRNA) thereof.

Protein

A protein of the present invention has an activity of the insulintranscription repressor belonging to the transcription factor CREMfamily, and has a function associated with nephropathy. Specifically,the protein has the functions associated with the onset and progression,and development of the symptom of nephropathy. The protein of theinvention particularly has the function associated with diabeticnephropathy in nephropathy.

The insulin transcription repressors include, for example, CREMα, ICERI, ICER Iγ, and the like. Among them, ICER Iγ is particularly useful.

A method for obtaining the protein of the invention is not particularlylimited, and for example, the protein can be obtained by the followingmethods.

An expression vector is constructed by inserting the gene of theinvention, e.g., the gene having the sequence described in SEQ ID NO:1or 2 ligated downstream of an appropriate promoter into the vector. Thenthe resulting expression vector is introduced into a host to produce atransformant. As the vector, for example, it is possible to useretrovirus type vector, papilloma virus vector, vaccinia virus vector,SV40 type vector, baculovirus vector, and the like. As the host, forexample, it is possible to use fungi, bacteria, yeast, insect cells,plant cells, animal cells and the like.

The nephropathy-associated protein of the present invention is obtainedby culturing the produced transformants and collecting the proteinproduced and accumulated in the culture. The produced protein can beisolated and purified by publicly known purification methods such assalting out by inorganic salts, fractionated precipitation by organicsolvent, ion-exchange resin column chromatography, affinity columnchromatography, gel filtration and immunoprecipitation. The protein canalso be formulated by publicly known methods.

It appears that the protein of the invention has strong relevance withmechanisms for the onset and progression of nephropathy, and thus, theprotein can be utilized effectively for the diagnosis of nephropathy,the analysis of the onset mechanism of nephropathy, determination ofmalignancy of nephropathy, and the like.

Antibody

An antibody of the present invention is an antibody which bindsspecifically to nephropathy-associated protein of the invention or apart thereof, can detect or measure the nephropathy-associated protein,and can be usefully utilized for the detection of nephropathy-associatedprotein, the diagnosis of nephropathy, the detection or determination ofnephropathy, the treatment of nephropathy and the like. The antibody ofthe invention can be suitably used, in particular, for diabeticnephropathy in nephropathy.

The antibody may be a polyclonal antibody or a monoclonal antibody aslong as it can bind specifically to the protein of the invention or apart thereof.

The monoclonal antibody is produced, for example as follows. Anindividual in which an antibody titer has been detected is selected fromnon-human mammalian animals, e.g., mice immunized with the protein ofthe present invention or a partial peptide thereof as an antigen, spleenor lymph nodes are removed 2 to 5 days after the final immunization, andantibody-producing cells contained therein are fused with myeloma cellsto prepare hybridomas which produce the monoclonal antibody. Themonoclonal antibody can be obtained by culturing the hybridomas toproduce the antibody and appropriately isolating/purifying the antibody.Cell fusion can be performed in accordance with known methods, e.g.,Kohler and Milstein's method (Nature, 256, 495, 1975) and modifiedmethods thereof (J. Immunol. Method, 39, 285, 1980; Eur. J. Biochem.,118, 437, 1981, Nature, 285, 446, 1980). As a fusion accelerator,polyethylene glycol (PEG) and Sendai virus and the like are used. Themonoclonal antibody of the invention may be used for humans by using anappropriate method known publicly.

A polyclonal antibody can be produced, for example as follows. Non-humanmammalian animals are immunized with the protein antigen itself or acomplex thereof with a carrier protein by the same method as in theproduction of the monoclonal antibody. The polyclonal antibody can beobtained by collecting an antibody against the protein of the inventionfrom the immunized animal and isolating/purifying the antibody.

By the use of the antibody of the present invention, it is possible todetect and measure nephropathy-associated protein. Also by utilizing theantibody of the invention, it is possible to detect, determine ordiagnose nephropathy (including diabetic nephropathy). For example, theonset, severity and prognosis of the nephropathy can be determined ordiagnosed by contacting the antibody of the invention with a sampleprepared from a specimen, and detecting or measuring an expressed amountor an expressed site of nephropathy-associated protein. In particular,the antibody of the invention can be utilized usefully for detection andtreatment of the diabetic nephropathy in nephropathy.

The antibody of the invention can also be appropriately formulated touse as a diagnostic, detecting or determining medicine of nephropathy ora preventive or therapeutic agent of nephropathy. The diagnosticmedicine of the nephropathy can also be utilized as a pharmaceutical fordetecting or measuring the nephropathy-associated protein. Thepreventive or therapeutic agent of the nephropathy can also be utilizedas a pharmaceutical for inhibiting the expression or the function of thenephropathy-associated protein. The pharmaceutical containing theantibody of the invention can be usefully utilized as the diagnosticmedicine of diabetic nephropathy or the preventive or therapeutic agentof diabetic nephropathy.

Preventive or Therapeutic Agent

The nephropathy-associated gene of the present invention can be involvedin the development of nephropathy, particularly diabetic nephropathy asthe complications of the diabetes. Therefore, a substance which inhibitsthe expression or the function of the gene is useful as not only thepreventive or therapeutic agent of nephropathy but also the preventiveor therapeutic agent of diabetic nephropathy and furthermore as thepreventive or therapeutic agent of the diabetes.

An inhibitor of the expression or the function of thenephropathy-associated gene, an antisense DNA of the gene and theantibody against the nephropathy-associated protein can be useful as thepreventive or therapeutic agent for the nephropathy, diabeticnephropathy and diabetes.

Transgenic Non-Human Mammalian Animal

A transgenic non-human mammalian animal in the present invention refersto a mammalian animal other than human, produced by gene recombinationby introducing the gene of the invention. The mammalian animals otherthan human include mice, rabbits, rats, and the like.

Methods for producing the transgenic non-human mammalian animal includea method (microinjection method) of directly introducing the gene in apronucleus of an ovum with a micropipette under a phase contrastmicroscope, a method of using an embryonic stem cell (ES cell), a methodof inserting the gene into a retrovirus vector or a adenovirus vector toinfect the ovum, and a sperm vector method by introducing the gene intothe ovum via a sperm.

In one preferable embodiment of the invention, a transgenic mouse (Tgmouse) of the nephropathy-associated gene (e.g., ICER, ICER Iγ)excessively expresses the gene specifically in β cells in pancreas. As aresult, the mouse develops the diabetes attributed to hyperglycemia,which can cause the nephropathy, i.e., the pure diabetic nephropathy.

The transgenic non-human mammalian animal of the invention exhibits thefollowing symptoms characteristic for the nephropathy.

-   1) A urinary volume, a urinary albumin amount and a urinary NAG    amount become large.-   2) Hypertrophy of renal pelvis is exhibited.-   3) Enlargement of renal tubules is exhibited.-   4) Early hypertrophy and fibrosing and subsequent sclerosis of    glomerulus are observed.

Furthermore, the transgenic non-human mammalian animal of the inventionexhibits the following symptoms characteristic of severe diabetes.

-   5) Polydipsia-   6) Polyuria-   7) Low body weight-   8) Hyperglycemia-   9) Low insulin

The most important symptoms in the transgenic non-human mammalian animalmodel of the present invention are the increase of the urinary albuminamount of 1) and the symptoms of 2) to 4) (the hypertrophy of renalpelvis, the enlargement of renal tubules, and the hypertrophy andfibrosing and subsequent sclerosis of glomerulus). In conventionaldiabetes models, although the increase of the urinary volume has beenobserved, no proteinuria has been observed. The proteinuria has beenobserved only in the double transgenic mouse, but its mechanism isconsidered to be quite different from the actual condition of thediabetes. In particular, the model which exhibits the symptoms(proteinuria and glomerular sclerosis) of the diabetic nephropathy asthe complications of the diabetes has been provided by the presentinvention for the first time.

Therefore, the transgenic non-human mammalian animal of the inventioncan be utilized usefully as not only an animal model for nephropathy butalso as an animal model for diabetes.

In particular, the transgenic non-human mammalian animal of theinvention is useful as the diabetic nephropathy model animal because theanimal develops the symptoms similar to those of the human diabeticnephropathy. For example, by the use of the transgenic non-humanmammalian animal of the invention, it is possible to perform screeningfor candidate substances of the diagnostic or therapeutic agent for thenephropathy or the diabetic nephropathy. Also by the use thereof, it ispossible to examine an appropriate dosage, a dosing period and potentialside effects of the candidate substance.

Screening

By applying the present invention, it is possible to efficiently performthe screening for the candidate substance of the diagnostic ortherapeutic agent for the nephropathy or the diabetic nephropathy.

For example, the candidate substances of the diagnostic or therapeuticagent for at least one selected from the group consisting of thenephropathy, the diabetic nephropathy and the diabetes can be screenedby administering a subject substance to the above transgenic non-humanmammalian animal of the invention and measuring an effect of the subjectsubstance on at least one selected from the group consisting of thenephropathy, the diabetic nephropathy and the diabetes of the mammaliananimal.

Substances effective for the treatment of nephropathy, diabeticnephropathy or the diabetes can also be screened using the expression ofa reporter gene as an indicator by adding the subject substance in theculture system of a human transformed cell line transformed with avector comprising the human transcription repressor, a promoter of, forexample, ICER Iy and the reporter gene ligated to be expressed by thepromoter.

A therapeutic or diagnostic medicine for the nephropathy or the diabeticnephropathy can be produced by appropriately formulating the substanceobtained by these screening methods as an active component or preparingit in combination with an appropriate pharmaceutically acceptablecarrier.

EXAMPLES

The present invention will be more specifically described with referenceto the following Examples, but the invention is not limited to theseExamples.

Example 1

(1) Materials and Procedures

Production of ICER Iγ Transgenic Mouse

An ICER Iγ cDNA was inserted downstream of a human insulin promoter in atransgenic plasmid plns-1. A transgene cassette (plns-ICER Iγ plasmid)was cut out with restriction enzymes, purified and introduced into afertilized ovum of C57BL/6×C57BL/6 (pure C57BL/6 mouse). A transgenicmouse (Tg) was identified by PCR (polymerase chain reaction) analysis ofa tail DNA using the following primers. Human insulin promoter, SEQ IDNO.1 5′-ATGGGCTCTGAGACTATAAAGCCAG-3′ (forward); Rabbit β globin, SEQ IDNO.2 5′-TGGATCCTGAGAACTTCAGG-3′ (forward 1); SEQ ID NO.35′-GCTGGTTATTGTGCTGTCTC-3′ (forward 2); ICER Iγ, SEQ ID NO.45′-CAGTTTCATCTCCAGTTACAGCCAT-3′ (reverse 1); and SEQ ID NO.55′-CTGCTTTATGGCAATAAGG-3′ (reverse 2);

A copy number of the transgene was examined by Southern blotting method.Littermates (wild type: WT) which were not transgenic mice were used inall experiments. All mice were handled in accordance with AnimalFacility Guideline at Kyoto University.

Measurement of Blood Glucose Value, Blood Parameters, Blood Pressure andHbAlc

A blood glucose value was measured in whole blood obtained from a tailby an enzyme-electrode method. The blood was quickly obtained from heartbefore the isolation of pancreas.

Levels of insulin, ketone and glucagon were measured using the followingELISA kits.

-   Insulin: MORINAGA Institute of Biological Science;-   ketone: Sanwa Kagaku; and-   glucagons: Yanaihara Institute Inc.

The blood pressure was measured using a tail cuff method.

The HbA1c value was measured using a DCA 2000 analyzer.

Isolation of Pancreatic Islets, Secretion and Content of Insulin

Pancreatic islets were isolated by a collagenase method. Insulinsecreting capacity, insulin content and DNA content were measured usingfresh pancreatic islets isolated from Tg (n=5) and WT (n=5) at 12 weeksof age by a batch incubation method, RIA and a fluorometric assay,respectively.

RNA Isolation, Reverse Transcriptase-Polymerase Reaction

Total RNA was extracted from the pancreatic islets freshly isolated fromTg (n=10) and WT (n=10) at 10 weeks of age using a trizol reagent (GibcoBRL). The presence of total islet RNA was identified by electrophoresis.A single strand c DNA was synthesized from the total islet RNA usingsuperscript reverse transcriptase. The expression of an ICER Iγ mRNAexpressed by the transgene was examined by a PCR method using the mouseislet cDNA and the following oligonucleotide primers. Rabbit β globin;5′-TGGATCCTGAGAACTTCAGG-3′ (forward; SEQ ID NO.2) and ICERIγ;5′-CTGCTTTATGGCAATAAGG-3′, (reverse; SEQ ID NO.5) Control β-actin;5′-ATCCGTAAAGACCTCTATGC-3′, (forward; SEQ ID NO.6)5′-AACGCAGCTCAGTAACAGTC-3′. (reverse; SEQ ID NO.7)

These primers are designed to stride an intron of the gene in order todistinguish the genomic DNA from the mRNA and confirm whether the DNA isnot contaminated at an RNA preparation stage. A Tg genomic DNAcontaining the intron and a WT islet cDNA having no transgene were usedas controls. The PCR was performed under a condition of 40 cycles of 94°C. for 15 seconds, 55° C. for 15 seconds, 72° C. for 30 seconds, and 72°C. for 5 minutes. PCR products were electrophoresed on 2% agarose geland stained with ethidium bromide.

Western Blotting

Western blotting was performed in order to examine the expression of anICER protein in the pancreatic islets of Tg. The pancreatic isletsisolated from Tg (n=3) and WT (n=3) at 10 weeks of age were homogenizedin a cell lysis solution (50 mM Tris-HCl(pH7.4), 150 mM NaCl, 1%TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 22 mM EDTA, 1% Trasilol),cell debris was removed by centrifugation, subsequently supernatantswere electrophoresed on 4 to 20% polyacrylamide SDS gel and transferredonto a polyvinylidene difluoride membrane in a transfer buffer (25 mMTris, 190 mM glycine, 20% methanol). The membrane was blocked with 5%skim milk in phosphate-buffered saline, and incubated with an anti-CREMantibody (diluted to 1:250; Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.) at 37° C. for 2 hours. Subsequently, the membrane was incubatedwith a second antibody, horseradish peroxidase-linked anti-rabbit IgG(Amersham Pharmacia Biotech) at 37° C. for 30 min. At these steps, themembrane was washed three times with 5% Tween 20/phosphate buffer for 10minutes. The detection was performed in accordance with ECL protocol(Amersham Pharmacia Biotech).

Northern Blotting

The total islet RNA (20 μg) was denatured in a solution of 1×MOPS(5×MOPS: 0.1 M MOPS, 40 mM sodium acetate, 5 mM EDTA), 6.7% formaldehydeand 50% formamide at 55° C. for 15 minutes, subsequently electrophoresedon 1.2% formaldehyde agarose gel and blotted onto a membrane. Afterprehybridization at 65° C. for one hour, hybridization with a ratinsulin cDNA probe was performed overnight. Both of the prehybridizationand the hybridization were performed in a solution of 5×sodiumchloride-sodium citrate(SSC), 0.1%sodium dodecyl sulfate at 65° C. Themembrane was washed twice with a solution of 0.1×SSC and 0.1% sodiumdodecyl sulfate at 65° C. for 30 minutes. Gene Images random primelabeling & detection system (Amersham Pharmacia Biotech) was used forlabeling and detection of the probe.

Urinary Volume, Urinary Albumin Amount and Urinary NAG Amount

The mice at 4 to 36 weeks of age produced as the above were individuallyhoused in a metabolic cage, and the urine was accumulated for 24 hours.The mice could eat and drink ad libitum during the accumulation ofurine. The amounts of urinary albumin and urinary NAG were measuredusing Alubuwell kit (Exocell Inc.) and NAG test kit (Shionogi),respectively.

Histological Analyses

(I) The kidney halves were fixed with a methyl Carnoy solution, embeddedin paraffin, and cut into serial sections (2 μm). Kidney sections werestained with PAS (periodic acid-Schiff) and PASM (periodicacid-methenamine silver), and observed by light microscopy. Theremaining kidney halves were snap-frozen in cold acetone in OCT compound, and cryostat sections were used for immunohistochemical staining.

A glomerular area was analyzed using the PAS staining. A sclerosis indexwas measured by scoring PAS-stained specimens (scores 0 to 3:0=notstained, 1=one-third or more, 2=one-third to two-thirds, 3=two-thirds ormore).

For fluorescent staining, the tissue was immediately embedded in an OCTcompound, and frozen in acetone-dry ice. This block was sectioned into3.5 μm sections using a cryostat. A rabbit polyclonal anti-mousecollagen type I antibody (Calbiochem), a rabbit polyclonal anti-mouselaminin antibody (Sigma), a rabbit polyclonal anti-mouse MMP-2 antibody(NeoMarks), and a rabbit polyclonal anti-mouse collagen type IV antibody(Progen Biotechnik) were used as primary antibodies. A FITC-labeled goatanti-rabbit antibody (Burlingame) was used as a second antibody.

(II) Parts of the removed kidney and pancreas were fixed with a Carnoysolution or 10% formalin, and subsequently embedded with paraffin. Theremainings were fixed with acetone, and subsequently embedded with OCT,which were then made into frozen sections (4 μm). Parts of kidneysections (3 μm) were used for PAS staining. Morphological analysis wasperformed by a blind test method using 6 to 8 kidneys at each week ofage. The following primary antibodies were used for immunologicalstaining. Anti-ICER Iγ antibody (1:500; provided by Dr. J. F. Habener,Massachusetts General Hospital, Howard Hughes Medical Institute, MA),anti-insulin antibody (1:500; DAKO, Kyoto, Japan), anti-collagen Iantibody (1:50; Calbiochem), anti-collagen IV antibody (1:250; Progen),anti-laminin antibody (1:100; Sigma), and anti-mouse MMP2 antibody(1:100; NeoMarks).

The primary antibody was fluorescently detected by a FITC-labeled secondantibody or a Texas red-labeled second antibody (1:200). Also afterdetection by a biotin-labeled second antibody, a DAB staining wasperformed. Stained sections were photographed using a confocalmicroscope (Zeiss LSM 410). The expressions of collagen I, collagen IVand laminin were semi-quantitatively analyzed by the blind test method.The following scores were used for the determination: 0=normal, 1=changeof 25% or less in the glomerulus, 2=change of 25 to 50%, 3=change of 50to 75%, and 4=change of 75% or more.

For electron microscopy, specimens were taken from the kidney of ICER Tg(n=4) and WT (n=4) mice at 40 weeks of age, fixed with 2% glutaraldehydein 0.05 mol/L phosphate buffer, dehydrated in a graded ethanol series,and embedded in epoxy resin according to routine procedures. Sectionswere cut with a glass knife on a Reichert-Nissei Ultracuts microtome.Sections were contrasted with uranyl acetate and lead citrate andobserved with electron microscope. Thickness of GBM was measured by theorthogonal intercept method.

(2) Data Analysis

The above data were represented as mean ±standard error (SE).Statistical comparison was performed using Student's t-test. A P valueless than 0.05 was considered to have a significant difference.

(3) Evaluation

Expression of ICER Iγ in Transgenic Mice

Effects of overexpression of ICER Iγ were evaluated using ICER Iγ Tgmice produced by the above method. A construct used is shown in FIG. 1a. The transgenic mice from three lines, i.e., Tg7, Tg12 and Tg23 wereused. The presence of ICER mRNA was identified by reverse transcriptasepolymerase chain reaction (RT-PCR) by amplifying sequences specific foroligonucleotides specific for rabbit β globin and ICER Iγ. A PCR productin which the intron had been spliced out and which corresponded to ICERIγ mRNA transcripted from the transgene was detected in ICER Iγ Tg mice(see FIG. 1 b). The Tg genomic DNA containing a fragment with intron andthe islet CDNA of the WT not containing the fragment were used as thecontrols. The expression of the ICER Iγ protein was identified by theanti-CREM antibody. It has been already reported that the anti-CREMantibody recognizes the ICER. As a result of performing the Westernblotting, it was found that the ICER Iγ was excessively expressed inICER Iγ Tg mice (see FIG. 1 c). Furthermore, the expression levels ofinsulin mRNA were analyzed using the islet total RNA extracted from ICERIγ Tg mice and the WT control mice at 10 weeks of age. The expressionlevel of the insulin mRNA was obviously lower in ICER Iγ Tg mice thanthe control mice (see FIG. 1 d).

Down regulation of the insulin mRNA corresponds to the expression ofICER Iγ at a high level, suggesting that the ICER Iγ is involved in thesuppression of insulin gene transcription activity.

Moreover, the number of the insulin-producing cells (β cells) has beenalready reduced on day 0, and the ICER Iγ directly causes the reductionof the β cells (FIG. 10A to D).

It is conceivable that the ICER Iγ excessively expressed in the β cellsinhibits the expression of tetracycline A which works importantly at aDNA synthesis phase in a cell cycle to inhibit proliferation of the βcells thereby reducing the number of the p cells in ICER Iγ Tg mice(FIG. 11).

Effects of High Level Expression of ICER Iγ

In order to evaluate the effects of the high level expression of ICERIγ, blood parameters were measured. ICER Iγ Tg mice developed severediabetes. Blood glucose levels in ICER Iγ Tg mice were normal on day 0,but increased markedly on day 7, increased further at 2 weeks of age,and remained at these high levels thereafter (see FIGS. 2 a and 2 b).Insulin concentrations in plasma were remarkably low and only one fifthof the control at 6 weeks of age (see FIG. 2 c). Ketone levels in bloodwere extremely high at 6 and 12 weeks of age, and further increaseduntil time of death (see FIG. 2 d). Glucagon levels in plasma were alsoremarkably high (see FIG. 2 e). In ICER Iγ Tg mice, ICER Iγ wasexpressed, and the number of the insulin-producing cell was reduced(FIG. 2 f).

The body weights of ICER Iγ Tg mice were similar to those of the controlon day 0 and day 7, but the body weights of ICER Iγ Tg mice were notincreased, and slightly increased after 8 weeks of age (see FIGS. 3 aand 3 c).

Then, the plasma parameters and the body weights were compared in malesand females of three transgenic lines, and the results are shown inTable 1. TABLE 1 Copy Body weight (g) Body glucose (mg/ml) Plasmainsulin (pg/ml) Line ♂♀ Number ♂ ♀ ♂ ♀ ♂ ♀ Control ♂ 0 33.2 ± 0.9  21.7± 0.7 153 ± 4.1    126 ± 9.9   1350.3 ± 194.5   1480.7 ± 194.5  Tg7 ♂ 425.8 ± 1.7^(a) 21.8 ± 0.6 527 ± 52.8^(a) 355 ± 61.7^(b) 350.5 ±80.0^(a)  422.4 ± 130^(a)  Tg12 ♂ 4 22.7 ± 2.6^(a) 22.5 ± 0.9 528 ±62.1^(a) 336 ± 93.2^(b) 545.6 ± 183.7^(b) 654.6 ± 84.2^(b) Tg23 ♂ 6 21.7± 2.4^(a) 22.6 ± 1.0 551 ± 31.1^(a) 420 ± 59.5^(b) 278.1 ± 111.2^(a)445.5 ± 39.7^(a)^(a)P < 0.05;^(b)P < 0.001 vs control.

Three lines (Tg7, Tg12 and Tg23) of ICER Iγ Tg mice with transgenepositive were established. The results are represented by mean ±SE of atleast 10 animals in each group. In either males or females at 12 weeksof age, only ICER Iγ Tg mice exhibited low insulin and hyperglycemia.The mice from Tg7 and Tg12 contain a different copy number of thetransgene from that of Tg23 mice, but no significant difference wasobserved in body weights, blood glucose levels, and insulin levels amongthe three lines. These results suggest that the specific expression ofICER Iγ at a high level is strongly linked to the symptoms of severediabetes.

Effects on Kidney

Furthermore, the effects on the kidney in ICER Iγ Tg mice wereevaluated. Urinary volumes were examined, and polyurea was detected inICER Iγ Tg mice (FIG. 4 a). Excretion of albumin in urine was examined,and high levels of proteinuria, which increased with age, was detectedin ICER Iγ Tg mice (FIG. 4 b). Urinary NAG, which was a marker of renaltubular disorder, also increased with age in ICER Iγ Tg mice (FIG. 4 c).Thus, urinary volume, urinary albumin and urinary NAG exhibited muchhigher values in ICER Iγ Tg than WT (control) mice.

Furthermore, WT and ICER Iγ Tg at 28 weeks of age were compared andanalyzed. These results are shown in Table 2. TABLE 2 28 wk WT Tg Weight(g) 33.7 ± 1.48 25.2 ± 1.44* Blood glucose (mg/dl) 140.3 ± 21.36 567.3 ±25.54* HbA1c (%)  4.0 ± 0.15 12.53 ± 0.77*  Blood Pressure 97.2 ± 1.59 114 ± 1.26* (mmHg) Serum creatinine  0.37 ± 0.007 0.39 ± 0.003 (mg/dl)Kidney/body weight 0.73 ± 0.04 1.69 ± 0.26* (%)In Table 2, * indicates significance at P < 0.05.

The amount of glycohemoglobin (HbA1c) was remarkably increased in ICERIγ Tg mice as compared with WT mice. Blood pressure was lower in ICER IγTg mice than in WT mice. Although the body weights of ICER Iγ Tg micewere lower than those of WT mice, a weight ratio of kidney/body weight(%) was considerably larger in ICER Iγ Tg mice than in WT mice.

Furthermore, the kidneys of ICER Iγ Tg mice were histologicallyexamined. As a result, swelling of kidney and enlargement of renalpelvis and tubules were observed (FIG. 5). In ICER Iγ Tg mice, thehypertrophy of glomerulus was observed at 4 weeks of age (FIG. 6), andsclerosis thereof was observed at 12 weeks of age. Obvious glomerularsclerosis was observed in ICER Iγ Tg mice at 36 weeks of age (FIGS. 7 a,7 b and 7 c). The glomerular hypertrophy was quantitatively examined,and the hypertrophy was observed from 4 to 12 weeks of age (FIG. 7 d).Also collagen type IV and laminin, which indicate fibrosing, wereincreased (FIGS. 8 and 9).

These are similar symptoms to those of human diabetic nephropathy, andindicate that the present invention is useful as the first diabeticnephropathy model mouse of the strain C57BL/6 which normally does notdevelop nephropathy.

By utilizing the gene or the protein of the invention, it is possible toeffectively diagnose or treat nephropathy, particularly diabeticnephropathy. For example, by the use of the probe based on the gene ofthe invention or the antibody which binds to the protein of theinvention, it is possible to diagnose or determine the onset of orelucidate the onset mechanism of nephropathy or diabetic nephropathy.

The transgenic non-human mammalian animal of the invention can beeffectively utilized as a proper evaluative model of nephropathy,particularly diabetic nephropathy, because it develops the symptomssimilar to nephropathy, particularly diabetic nephropathy, in humans.

Furthermore, by the use of the transgenic non-human mammalian animal ofthe invention, it is possible to efficiently perform the determinationor diagnosis of nephropathy or diabetic nephropathy, or the screeningthe active component of the preventive or therapeutic agent ofnephropathy or diabetic nephropathy.

The present invention provides exceptionally excellent procedures in thediagnosis or the treatment of nephropathy or diabetic nephropathy.

1. A nephropathy-associated gene comprising a nucleic acid encoding aninsulin transcription repressor belonging to a transcription factor CREMfamily.
 2. The gene according to claim 1 wherein the insulintranscription repressor is selected from the group consisting of CREMα,ICER I and ICER Iγ.
 3. The gene according to claim 1 wherein the insulintranscription repressor is ICER Iγ.
 4. The gene according to claim 1having a base sequence represented by SEQ ID NO: 1, a complementarysequence thereof or a nucleotide sequence which hybridizes therewithunder a stringent condition, and capable of developing nephropathy. 5.The gene according to claim 1 having a base sequence represented by SEQID NO:2, a complementary sequence thereof or a nucleotide sequence whichhybridizes therewith under a stringent condition, and capable ofdeveloping nephropathy.
 6. The gene according to claim 1 wherein thenephropathy is diabetic nephropathy.
 7. The gene according to claim 1wherein said gene is derived from human.
 8. A nephropathy developingagent comprising the gene according to claim
 1. 9. A diabeticnephropathy developing agent comprising the gene according to claim 1.10. A probe for determination or diagnosis of nephropathy comprising 15or more bases of nucleotide sequence capable of hybridizing with thegene according to claim 7 under a stringent condition.
 11. Anephropathy-associated protein having an activity of an insulintranscription repressor belonging to a transcription factor CREM family.12. The nephropathy-associated protein according to claim 11 which isselected from the group consisting of CREMα, ICER I and ICER Iγ.
 13. Theprotein according to claim 11 derived from human.
 14. An antibody whichspecifically binds to the protein according to any of claim 11 or a partthereof.
 15. The antibody according to claim 14 which specifically bindsto the human nephropathy-associated protein.
 16. A diagnostic medicineof human nephropathy containing the antibody according to claim
 15. 17.An antisense DNA for the human nephropathy-associated gene according toclaim
 7. 18. A preventive or therapeutic agent of neuropathy comprisingthe antibody according to claim 15 or the antisense DNA according toclaim 17 as an active component.
 19. A method for determining ordiagnosing nephropathy comprising a step of examining an expressionlevel of the human nephropathy-associated gene according to claim 7 in aspecimen using the probe according to claim 10 or the antibody accordingto claim
 12. 20. A transgenic non-human mammalian animal comprising thegene according to claim
 1. 21. The mammalian animal according to claim20 that develops nephropathy.
 22. The mammalian animal according toclaim 21 wherein the nephropathy is diabetic nephropathy.
 23. Themammalian animal according to claim 20 that develops diabetes.
 24. Themammalian animal according to claim 23 wherein said diabetes combineswith diabetic nephropathy.
 25. A method for making a transgenicnon-human mammalian animal comprising a step of introducing the geneaccording to claim
 1. 26. A method for screening a preventive ortherapeutic agent for at least one selected from the group consisting ofnephropathy, diabetic nephropathy and diabetes, comprising a step ofadministering a subject substance to the transgenic non-human mammaliananimal according to claim 20 and measuring an effect of the subjectsubstance on at least one selected from the group consisting ofnephropathy, diabetic nephropathy and diabetes of the mammalian animal.27. An expression vector capable of stably expressing in mammaliancells, comprising a marker gene under the control of a promoter of thehuman nephropathy-associated gene according to claim
 7. 28. A mammaliancell comprising the expression vector according to claim
 27. 29. Thecell according to claim 28 wherein the mammalian cell is a human cell.30. A method for screening a preventive or therapeutic agent for atleast one selected from the group consisting of human nephropathy,diabetic nephropathy and diabetes, comprising a step of culturing themammalian cells according to claim 28 or in the presence of a medicinecandidate compound in culture of the mammalian cells and a step ofmeasuring an amount of the expressed marker gene in the presence orabsence of the candidate compound.
 31. A preventive or therapeutic agentfor at least one selected from the group consisting of nephropathy,diabetic nephropathy and diabetes, comprising a substance obtained bythe method for screening according to claim 26 or 30 as an activecomponent.